We have shown a severe decrease in ATP-dependent acidification of endosomes isolated from a Chinese hamster ovary cell mutant pleiotropically defective in receptor-mediated endocytosis. This mutant DTG 1-5-4, and many other of our endocytosis mutants, also exhibited loss of some Golgi-associated functions: in DTG 1-5-4 asparagine-linked oligosaccharides on Sindbis virus glycoproteins were not galactosylated and Sindbis virus assembled on Golgi cisternae rather than on the plasma membrane; also, some endogenous glycoproteins showed incomplete terminal glycosylation. Completion of glycosylation in the mutant seems dependent on the nature of the glycoprotein acceptor: glycoprotein G of vesicular stomatitis virus was galactosylated although Sindbis glycoproteins were not; most cell-associated glycoproteins were glycosylated normally but secreted glycoproteins were not. In DTG 1-5-4 defects in endocytosis, endosomal acidification and Golgi function result from a single genetic lesion since these activities were all restored in revertants of DTG 1-5-4 and on genetic complementation. To determine if impairment of Golgi-associated post-translational modification in the mutants resulted secondarily in defective endocytosis, we have examined a temperature-sensitive mutant. After a shift to non-permissive temperature (39 C) endocytic activities were lost prior to detectable changes in post-translational modification. Also, endosomes isolated from the mutant grown at the permissive temperature (34 C) exhibited ATP-dependent acidification at 34 C, but not at 39 C. Thus, the endosomal defect is not secondary to the Golgi defect; we suggest that endosomes and Golgi require a common component, defective in the mutants. We are presently attempting to identify this component.